We are studying the mechanism of negative control of gene regulation at the level of transcription using the genes of D-galactose utilization in Escherichia coli. We have demonstrated that at least five unlinked transcriptional units on the bacterial chromosome constitute a regulon (gal regulon). Two of the genes (galR and galS) encode two negative regulators, two others are responsible for the high (mgl operon) and low affinity (galP) galactose transport, and the fifth one (gal operon) encodes the synthesis of galactose metabolizing enzymes. Four of the transcription units contain two operators, OE and OI. The mgl operon contains only OE. GalR and GalS, which have 85% amino acid sequence similarities, bind to the same operator(s), but do not regulate each member of the regulon in a parallel manner. GalS also autoregulates its own gene. We have isolated and characterized three Gal repressor mutants, called Gal super-repressor (GalRs), which are noninducible in the presence of inducer galactose. The mutational changes have been located to a region of the gene which we have previously assigned to be part of the sugar binding domain by sequence comparison of similar proteins. Biochemical analysis of the mechanism of negative control in a purified system has shown that: (i) Complete repression of the two promoters of the gal operon requires an interaction between the OE and OI bound repressor molecules which generate a DNA loop encompassing the promoter segment; (ii) RNA polymerase remains bound to gal promoter DNA under conditions of DNA looping; (iii) Binding of repressor to OE alone causes partial repression of P1 or P2 if its location is separated from OE by an integral number of DNA helical turns. From these results we have proposed that repressor does not repress the gal operon by inhibiting RNA polymerase binding but locks the promoter-bound RNA polymerase in an inactive state. Repressor bound to OE makes a direct contact to inhibit transcription. Such inhibition is further aided by forming a DNA loop around the surface of the RNA polymerase.